α tubulin mouse monoclonal antibody

Cells at 70% to 80% confluence were washed twice with ice-cold phosphate-buffered saline, lysed on ice in radioimmune-precipitation assay buffer (RIPA, Cell Signaling Technology, Danvers, MA) containing a complete protease inhibitor cocktail (Roche Applied Sciences, Mannheim, Germany), and then heated for 5 minutes at 100°C. Fresh tissue samples were ground to powder in liquid nitrogen and lysed with sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer. Briefly, equal amounts of protein (30 μg) were separated by electrophoresis on a 10.5% sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Immobilon P, Millipore, Bedford, MA). After blocking with 5% milk solution in Tris-buffered saline with Tween-20 (TBST) for 1 hour, the membranes were incubated with primary antibody using anti-PTOV1 antibody (1:100, Sigma, HPA051812) overnight at 4°C. α-Tubulin mouse monoclonal antibody (1:1000, Santa Cruz Biotechnology) was used as an internal loading control. After 3 washes with TBST, the membranes were incubated with secondary antibodies against rabbit immunoglobulin G or mouse immunoglobulin G. PTOV1 expression was then examined using the enhanced chemiluminescence detection system (Amersham Biosciences Europe, Freiberg, Germany) according to the manufacturer's instructions.